1. Field of the Invention
The present invention relates to a reference electrode for use in a continuous flow type measuring apparatus incorporating an immobilized enzyme electrode and, more particularly, to a silver/silver chloride reference electrode which eliminates the possibility of enzyme activity being inhibited due to silver ion run off.
In this specification the term "hydrophilic gel" means a water-containing gel which has affinity for water but is insoluble in water, and which allows migration of ions; it includes no hydrophobic gel.
2. Description of the Prior Art
Measuring techniques using enzyme electrodes for measurement of various chemical substances have recently been widely employed in such areas as clinical chemistry and food chemistry, since they provide a combination of the substrate specificity of enzymatic reaction and the characteristic features of electrochemical analysis such as measurement fastness and high measurement sensitivity. Measuring techniques utilizing enzyme electrodes are classified into the following two kinds of methods:
(1) potentiometric method (or a method of measurement through potential difference detection); and
(2) amperometric method (or a method of measurement through detection of electrolytic current at controlled potential). In the potentiometric method, it is necessary that the potential of the working electrode to the reference potential generated at the reference electrode must be accurately measured; and in the amperometric method, it is essential that the potential of the working electrode must be accurately maintained.
In order to accomplish such accurate measurement, the potential of the reference electrode, as a reference potential, must be kept stable, and further the reference electrode must meet the following requirements:
(I) that the electrode reaction on the surface of the reference electrode is reversible and is responsive to a particular chemical species in the electrolyte according to the Nernst equation;
(II) that the reference electrode provides the potential of the reversible reaction, that is stable over a long-time and highly reproducible;
(III) that in the amperometric method wherein two electrodes are employed, the potential of the reference electrode is not changeable, at least only to a negligible extent, due to current during the measuring operation (i.e., non-polarizable) and, when there is any potential change due to any slight current flow, the potential quickly reverts to its original level; and
(IV) that there is no potential hysteresis with respect to temperature changes.
Silver/silver chloride reference electrodes are known as satisfying these requirements. A silver/silver chloride reference electrode comprises a half cell consisting of a silver electrode covered with a silver chloride containing layer and a chlorine ion-containing internal solution; and the internal solution of the electrode is electrically connected through a suitable liquid junction to an electrolytic solution to be analyzed. The liquid junction must meet the following requirements:
(I) that in view of the fact that, while the flow rate of the internal solution from the reference electrode should not be unreasonably large in order to minimize contamination of the solution to be analyzed by the internal solution, an excessively slow flow of the internal solution may result in the occurrence of an abnormal liquid junction potential. Therefore, the liquid junction should allow a moderate flow of the internal solution;
(II) that in order to obtain a stable and well reproducible liquid junction potential, the flow rate of the internal solution should be constant.
That is, a reference electrode should be such that the internal solution is allowed to flow at a constant rate out of the electrode through the liquid junction.
To this end, micro-pore, salt bridges, fritted glass means, or the like have hitherto been employed as liquid junctions that electrically interconnect the reference electrode and the electrolytic solution for electrolytic measurement.
The miniaturization of a reference electrode makes it possible to reduce the distance between the working electrode and the reference electrode, thereby lowering the degree of possible voltage drop due to solution resistance, so that the potential of the working electrode can be kept more stable, it being thus possible to obtain very accurate measurements. Therefore, attempts have been made to reduce the size of silver/silver chloride reference electrodes; however, the attempts have involved such problems as are discussed below.
The miniaturization of a silver/silver chloride reference electrode along with the reduction of size of a relevant enzyme electrode results in an increased proportion of the internal solution flowing out through the liquid junction in relation to the total amount of the internal solution, which fact possesses a problem with respect to the potential stability of the reference electrode.
Another problem is that when the size of the liquid junction for the silver/silver chloride reference electrode is reduced to slow down the flow of the internal solution through the junction, an abnormal liquid junction potential is likely to occur at the liquid junction.
Further problems are that where salt bridges are employed, it is impracticable to miniaturize the whole of the silver/silver chloride reference electrode including the salt bridges, and that if there is any difference in chlorine ion concentration between the internal solution of the electrode and the solution being measured, it is difficult to obtain good potential stability because the internal solution is liable to change in chlorine ion concentration.
As pointed out above, the prior art silver/silver chloride reference electrodes having an internal solution and a liquid junction can hardly be miniaturized because of their structural configuration; and therefore, the present inventors have considered a concept that a reference electrode having a silver chlorde containing layer provided on silver wires is directly inserted into electrolytic solution for electrolytic measurement.
A silver/silver chloride reference electrode is produced, for example, by the following. A silver electrode is dipped into the electrolytic solution containing chlorine ions. Electric current is flowed between the silver electrode as anode and a platinum electrode by employing, for example, a controlled current generating apparatus, whereby a thin silver/silver chloride layer is formed on the surface of the silver electrode (electroplating method). This method has an advantage that it can easily produce silver/silver chloride electrodes, on the one hand, but on the other hand, it involves disadvantages in that the thin layer formed is not satisfactory in physical strength, and that the thin layer is likely to peel off in the course of prolonged use, it being thus unable to obtain good potential stability.
A reference electrode for an enzyme electrode which is press-molded from a silver/silver chloride/silver sulfide mixture is also known (Japanese Patent Laid Open Publication No. 43556/1987). This reference electrode has the drawback that production of the electrode requires difficult molding work, which fact virtually denies the possibility of such electrode being produced in a miniaturized form.
When, as above described, a silver/silver chloride reference electrode is placed in the electrolytic solution directly and without use of any salt bridge, an enzyme electrode may, depending upon the variety of enzymes used therein, lose their activity very rapidly due to silver ions dissolved slightly from the silver/silver chloride reference electrode.
Silver chloride is slightly soluble in water (1.9 mg/l), and silver ions enter into good combination with proteins. Whilst, certain enzymes may be adversely affected or inactivated by the presence of silver ions. A number of such enzymes are known including, for example, L-sorbose oxidase (E. C. 1, 1, 3, 11), inulinase (E. C. 3, 2, 1, 7), .alpha.-D-glucosidase (E.C. 3, 2, 1, 20), .beta.-D-glucosidase (E. C. 3, 2, 1, 21), .beta.-D-galactosidase (E.C. 3, 2, 1, 23), and invertase (E. C. 3, 2, 1, 26).
It has been proposed to employ a palladium electrode as a reference electrode in order to prevent inactivation of enzymes due to silver ions (as in U.S. Pat. No. 4,547,280), but such electrode does not fully satisfy the aforesaid requirements for reference electrodes; it still involves a problem from the view point of long-term potential stability.
It is apparent from what is discussed above that no effective measures have ever been proposed for miniaturization of silver/silver chloride reference electrodes.